Transposon mutagenesis reveals cooperation of ETS family transcription factors with signaling pathways in erythro-megakaryocytic leukemia

Jian Zhong Tang; Catherine L. Carmichael; Wei Shi; Donald Metcalf; Ashley P. Ng; Craig D. Hyland; Nancy A. Jenkins; Neal G. Copeland; Viive M. Howell; Zhizhuang Joe Zhao; Gordon K. Smyth; Benjamin T. Kile; Warren S. Alexander

doi:10.1073/pnas.1304234110

Contributed by Donald Metcalf, March 4, 2013 (sent for review January 30, 2013)

Abstract

To define genetic lesions driving leukemia, we targeted cre-dependent Sleeping Beauty (SB) transposon mutagenesis to the blood-forming system using a hematopoietic-selective vav 1 oncogene (vav1) promoter. Leukemias of diverse lineages ensued, most commonly lymphoid leukemia and erythroleukemia. The inclusion of a transgenic allele of Janus kinase 2 (JAK2)V617F resulted in acceleration of transposon-driven disease and strong selection for erythroleukemic pathology with transformation of bipotential erythro-megakaryocytic cells. The genes encoding the E-twenty-six (ETS) transcription factors Ets related gene (Erg) and Ets1 were the most common sites for transposon insertion in SB-induced JAK2V617F-positive erythroleukemias, present in 87.5% and 65%, respectively, of independent leukemias examined. The role of activated Erg was validated by reproducing erythroleukemic pathology in mice transplanted with fetal liver cells expressing translocated in liposarcoma (TLS)-ERG, an activated form of ERG found in human leukemia. Via application of SB mutagenesis to TLS-ERG–induced erythroid transformation, we identified multiple loci as likely collaborators with activation of Erg. Jak2 was identified as a common transposon insertion site in TLS-ERG–induced disease, strongly validating the cooperation between JAK2V617F and transposon insertion at the Erg locus in the JAK2V617F-positive leukemias. Moreover, loci expressing other regulators of signal transduction pathways were conspicuous among the common transposon insertion sites in TLS-ERG–driven leukemia, suggesting that a key mechanism in erythroleukemia may be the collaboration of lesions disturbing erythroid maturation, most notably in genes of the ETS family, with mutations that reduce dependence on exogenous signals.

The study of cancer-causing genes using insertional mutagenesis in the mouse (1, 2) provides a powerful complement to human cancer genomics for functional identification and characterization of the genetic lesions driving tumor development. Transposons are DNA elements with the unique capacity to change their genomic position, usually via expression of a transposase, an enzyme that catalyzes excision of the transposon from the genome and facilitates its reintegration elsewhere. Although most mammals lack active endogenous transposons, the Sleeping Beauty (SB) system has been developed recently to adapt the well-studied fish Tc/mariner transposon to allow insertional mutagenesis in the mouse (2). Temporally controlled or tissue-specific SB-mediated genetic screens have been designed by imposing cre-recombinase control over the expression of the transposase (2). Moreover, by activating transposition in mice carrying a predisposing germ-line genetic lesion, insertional mutations that cooperate with that particular lesion in cancer pathogenesis can be isolated specifically.

To define genetic lesions driving leukemia, we targeted SB transposon mutagenesis to the blood-forming system by intercrossing mice transgenic for both a transposon array and a cre-inducible SB transposase allele (3) with mice expressing cre recombinase from the hematopoietic-selective vav 1 oncogene (vav1) promoter (4). Leukemias of diverse cellular origins developed in the ensuing offspring, most commonly erythroleukemia and lymphoid leukemia. In the presence of a transgene expressing Janus kinase 2 (JAK2)V617F (5), an active form of JAK2 associated with hematological disease (6), accelerated transposon-driven disease ensued with selection for erythroleukemic pathology characterized by transformation of bipotential erythro-megakaryocytic cells. The genes encoding the E-twenty-six (ETS) transcription factors Ets related gene (Erg) and Ets1 were common sites for transposon insertion (CIS) in the JAK2V617F-positive leukemias. We validated the role of Erg by reproducing the erythroleukemic pathology in mice transplanted with hematopoietic cells expressing translocated in liposarcoma (TLS)-ERG, an activated form of ERG found in human leukemia (7). SB mutagenesis in TLS-ERG–induced leukemia identified Jak2 as a CIS, together with other loci expressing regulators of signal transduction, suggesting that a key mechanism in erythroleukemia may be the collaboration of lesions disturbing erythroid maturation, most notably in genes of the ETS family, with mutations that reduce dependence on exogenous signals.

Results

Mice homozygous for both a transposon array and a cre-inducible SB transposase allele (T2/Onc2/T2/Onc2;RosaSB^LSL/RosaSB^LSL) (3) were mated with mice hemizygous for independent transgenes allowing expression of cre (4) and JAK2V617F (5) from the vav1 promoter (vav-cre/+;JAK2^V617F/+; Fig. S1A). Mice of each of the four resultant genotypes [transposon-active, with or without the JAK2V617F transgene (T2Onc2/+;SB^LSL/+;vav-cre/+;JAK2^V617F/+ and T2Onc2/+;SB^LSL/+;vav-cre/+, referred to as JAK2SB^vav and SB^vav), as well as transposon-inactive controls (T2Onc2/+;SB^LSL/+;JAK2^V617F/+ and T2Onc2/+;SB^LSL/+, referred to as JAK2 and SB^LSL)] were weaned in numbers expected from normal Mendelian segregation of alleles (JAK2SB^vav:SB^vav:JAK2:SB^LSL = 79:69:70:75). To examine the efficiency of activation of SB transposase, we analyzed expression of GFP, which is present before excision but deleted upon cre-mediated recombination of the conditional allele (3). Loss of GFP positivity was observed in all of six SB^vav mice analyzed. In two of these mice, recombination was evident in effectively all hematopoietic cells examined whereas, in the others, efficiency varied from 30% to 80%. Although interanimal variation was observed, in any individual mouse, the efficiency of recombination was consistent among hematopoietic cells of different lineages and maturation stages (Fig. S1B).

JAK2V617F Accelerates SB-Induced Erythroleukemia.

As expected, SB^LSL mice displayed no significant illness and, whereas the presence of the JAK2V617F transgene has been shown to cause increased numbers of circulating blood cells (5), these mice also remained healthy (Fig. 1A). In contrast, activation of SB transposition in SB^vav mice resulted in disease in all mice within 12 mo, with 50% affected within 100 d. Consistent with activation of transposition in multiple hematopoietic lineages, histopathological analysis revealed examples of lymphoid, myeloid, megakaryocytic, and erythroid disease in SB^vav mice although the majority of mice developed lymphoid malignancy, erythroleukemic pathology, or both (Fig. 1B). Strikingly, the presence of the JAK2V617F transgene resulted in significantly accelerated disease: all but 1 of 63 JAK2SB^vav mice succumbed to disease within 70 d, and most displayed erythroleukemic pathology, occasionally coincident with lymphoid disease (Fig. 1B)

Fig. 1.

(A) Survival of SB^LSL (n = 23), JAK2 (n = 27), SB^vav (n = 48), and JAK2SB^vav (n = 63) mice. Time indicates age at intervention due to illness. P < 0.0001 for comparison of JAK2SB^vav mice with all other genotypes and SB^vav mice with SB^LSL controls. (B) Spectrum of leukemias developing in SB^vav and JAK2SB^vav mice. The sector size in the pie chart represents the proportion of mice that developed leukemia of the indicated cellular origin based on histopathological examination. In some mice, two distinct leukemia types were evident. (C) Histological presentation of erythroleukemic pathology in representative JAK2SB^vav mice showing infiltration of bone marrow and spleen with nucleated erythroid cells.

In SB^vav mice, the lymphoid leukemias typically involved the bone marrow, spleen, thymus, and lymph nodes (Fig. S2), with or without a significant elevation in circulating white blood cells. The disease displayed a consistent histological appearance, with affected organs containing tightly packed blast cells interspersed with phagocytic macrophages and often the appearance of free aggregates of leukemic cells within the peritoneal cavity fatty tissue. Flow cytometric analysis confirmed the lymphoid origin of disease and established that the leukemic cells were T-lymphoid, either CD4/CD8 double negative or single positive.

In both SB^vav and JAK2SB^vav mice, the erythroleukemic pathology included consistently high peripheral blood nucleated cell counts dominated by nucleated erythroid cells, which were often accompanied by thrombocytopenia. The mice typically presented with a grossly enlarged spleen dominated by a population of immature nucleated erythroid cells and little surviving normal spleen architecture. The bone marrow was usually infiltrated by similar erythroid cells, as was the liver, with hepatomegaly resulting from the uniform distribution of these cells throughout the organ. Thickening of the lung alveolar walls by infiltrating nucleated erythroid cells was also commonly observed (Fig. 1C and Fig. S3).

JAK2SB^vav Leukemia Is Transplantable.

Suspensions of bone marrow and/or spleen cells from five erythroleukemic JAK2SB^vav mice were injected into sublethally irradiated recipient mice. All recipients succumbed to disease 13–31 d after transplantation (Table S1), and, in all cases examined, leukemia was evident, with the same characteristics as that observed in primary mice: thrombocytopenia and high nucleated blood cell counts, as well as splenomegaly and hepatomegaly resulting from infiltration of immature nucleated erythroid cells.

In methylcellulose cultures, spleen cells from primary JAK2SB^vav leukemias failed to generate colonies in the absence of cytokine stimulation and produced small numbers of colonies in the presence of stem cell factor (SCF) and/or IL-3. Larger numbers of colonies developed in cultures containing erythropoietin (EPO), suggesting that the leukemic cells were primarily dependent on EPO, and the combination of EPO with SCF and/or IL-3 yielded maximal colony numbers (Table S2). A similar result was observed in cultures of secondary leukemias: whereas in three of five cultures some colonies developed in the absence of exogenous cytokine, the majority of clonogenic cells remained cytokine dependent (Table S2). The colonies observed were large and compact (Fig. 2A) and, when picked and stained, were remarkably observed to contain nucleated erythroblasts and acetylcholinesterase-staining megakaryocytic cells (Fig. 2A). The presence of both erythroblasts and megakaryocytic cells was confirmed by flow cytometric analysis of cells from individual colonies. All cells in each colony were CD71⁺ Ter119^−/lo, and a proportion also expressed CD41 (Fig. 2B). Individual colonies contained varying numbers of megakaryocytic cells, from just a few percent up to the majority of cells (Fig. 2C), but this observation was a consistent feature of almost all leukemias examined, suggesting that the transformed cells in JAK2SB^vav leukemias were in fact bipotential. Thus, although the megakaryocytic component of the leukemias was not readily evident in most histological sections, based on these observations, we classified the disease as erythro-megakaryocytic leukemia.

Fig. 2.

(A) Morphology of colonies in methylcellulose cultures of spleen cells from a representative JAK2SB^vav mouse (Top). Cytocentrifuge preparations from a single colony stained with megakaryocyte-specific acetylcholinesterase (AChE) plus Luxol fast blue/hematoxylin revealing characteristic red-stained megakaryocytes (Middle) or May Grunwald Giemsa showing erythroid cells (Bottom). (B) Flow cytometric profiles of cells pooled from JAK2SB^vav colonies showing costaining of CD71⁺ Ter119^−/lo with CD41 in a proportion of cells. (C) Frequency of megakaryocytic cells in consecutively picked colonies from cultures of JAK2SB^vav spleen cells. Individual colonies contained varying numbers of AChE⁺ megakaryocytes, from a few percent up to the majority of cells, but this observation was a consistent feature in 12 of the 13 mice examined.

Common Transposon Insertion Sites in JAK2SB^vav Leukemia.

The transposon integration sites in spleen cells from 40 JAK2SB^vav mice with erythro-megakaryocytic leukemia (7 with concurrent lymphoid leukemia) were isolated via linker-mediated PCR incorporating barcoded primers and identified by pyrosequencing. CIS were identified using bioinformatic strategies outlined in Materials and Methods. From the 40 samples, a total of 247,502 sequence reads were obtained, of which 112,888 (46%) were successfully mapped to the mouse genome.

Twenty-three CIS were identified in the JAK2SB^vav leukemias (Table 1). The most frequently identified were loci encoding members of the ETS family of transcription factors: Erg and Ets1, which were identified in 35 (87.5%) and 26 (65%) leukemias, respectively. In 22 (55%) samples, transposon integration at both Erg and Ets1 was evident. Analysis of the integration sites at the Erg locus revealed a uniform transposon orientation and clustering of insertion sites within introns 3 and 4 (Fig. 3A), consistent with expression of a transposon-Erg fusion transcript regulated by the T2/Onc LTR/splice donor elements (2) and reminiscent of the mode of ERG activation caused by intragenic translocations in prostate cancer and leukemia (7, 8). All of the transposon integrations within the Ets1 gene clustered upstream of exon 1 or within the first intron and were uniformly oriented in a manner indicative of transposon-activated expression of a full-length transcript or a fusion transcript excluding the first exon (Fig. 3A). Of note, in several individual leukemias, multiple transposon integration sites were identified within Erg or Ets1, and this feature may reflect ongoing transposition during leukemia development. Loci previously implicated in hematopoietic malignancies were also prominent, including Vav1, zinc finger, MIZ-type containing 1 (Zmiz1), and BCL6 interacting corepressor (Bcor) (Discussion).

View this table:

Table 1.

Common transposon insertion sites in JAK2SB^vav erythroleukemic mice

Fig. 3.

(A and B) Arrowheads indicate the sites of transposon integration within the genes encoding the ETS family proteins Erg and Ets1 in JAK2SB^vav leukemias (A) and within Jak2 in TLS-ERG–induced leukemias (B), with the direction indicating transposon orientation. Exons are numbered and shown as raised boxes. (C) CIS were examined for common cellular processes or pathways using a combination of GO term analysis and literature searching.

Identification of Cooperating Lesions in ERG-Driven Leukemia.

To validate the leukemia-causing potential of CIS identified in JAK2SB^vav mice, wild-type fetal liver cells were infected with a retrovirus expressing GFP plus TLS-ERG, a leukemia-derived product of intragenic translocation of ERG, chosen to mimic the activation of Erg via intragenic transposon insertion, and then transplanted into myeloablated recipient mice (Fig. 4A). Twenty-five of 27 transplanted mice developed disease (Fig. 4B), and, of 24 mice examined, 22 displayed erythroleukemic pathology. Three of these mice also had evidence of a concurrent lymphoid leukemia; however, the lymphoid component was derived from uninfected (GFP⁻) cells and likely reflected co-occurrence of TLS-ERG–induced erythroid disease and radiation-induced lymphoid leukemia. The leukemia in recipients of TLS-ERG–transduced fetal liver cells was notably similar to that observed in JAK2SB^vav mice, with splenomegaly caused by excess immature nucleated erythroid cells, infiltration of the bone marrow, liver, and lungs by immature erythroid cells, and an abnormal population of CD71 Ter119^−/lo cells in the bone marrow, spleen, and blood of the affected mice. Expression of significant levels of CD41 was also observed in two of seven leukemias examined.

Fig. 4.

(A) SB transposon mutagenesis in TLS-ERG–driven leukemia. Mice homozygous for a T2/Onc transposon array were mated with mice constitutively expressing SB transposase (RosaSB/RosaSB), and fetal liver cells were infected with retroviruses expressing TLS-ERG-GFP or control GFP and transplanted into wild-type recipient mice. (B) Survival curves for recipients of WT-GFP (n = 14), SB-GFP (n = 17), WT-TLS-ERG (n = 27), and SB-TLS-ERG (n = 34) fetal liver cells. Time indicates age at intervention due to illness. P < 0.0001 for comparison of survival of SB-TLS-ERG mice with that of SB-GFP or WT-TLS-ERG cohorts.

To explore the multistep nature of leukemogenesis specifically involving activation of Erg, fetal liver cells carrying a transposon array and constitutively expressing SB transposase were infected with the TLS-ERG retrovirus. Consistent with cooperation between Erg activation and alleles affected by transposon insertion, disease developed significantly more rapidly in recipients of TLS-ERG–transduced SB fetal liver compared with recipients of TLS-ERG–transduced wild-type cells, or control GFP-transduced SB cells (Fig. 4B). Indicative of a particular role for Erg in driving erythroid transformation, whereas control GFP-transduced SB cells yielded leukemias of diverse origins in recipient mice (six lymphoid, three erythroid, one lymphoid/erythroid, and two myeloid), all but one recipient of SB TLS-ERG cells developed eythroleukemic pathology indistinguishable from that observed in recipients of TLS-ERG-transduced wild-type cells (Fig. S4). A minority of SB TLS-ERG recipients developed GFP⁻ lymphoid or erythroid leukemias, indicative of SB-only–driven disease.

Twenty-five CIS encompassing 28 genes were identified by analysis of 13 recipients of TLS-ERG–transduced SB cells, all of which showed erythroleukemic pathology (Table 2). The Jak2 locus was identified as a CIS in six independent samples (Fig. 3B), strongly validating the cooperation between JAK2V617F and transposon insertion at the Erg locus in JAK2SB^vav leukemia. To explore pathways cooperating with Erg in erythroid transformation, CIS that co-occurred with Erg in JAK2SB^vav mice or were identified in TLS-ERG–driven leukemias were examined for common cellular processes or pathways using a combination of Gene Ontology (GO) term analysis and literature searching. Transcription factors and epigenetic regulators of gene expression were present, as were genes involved in stem cell pluripotency and cell migration and adhesion; however, particular enrichment was observed for growth factors, receptors, and regulators of signal transduction (Fig. 3C and Discussion).

View this table:

Table 2.

Common transposon insertion sites in TLS-ERG-induced erythroleukemic mice

Discussion

The molecular changes associated with human acute erythroleukemia (AEL) are poorly characterized (9, 10). The leukemias in JAK2SB^vav mice provided an opportunity to define molecular lesions driving erythroid transformation via identification of transposon integration sites. Of note, the two most frequently observed CIS in JAK2SB^vav erythroid leukemias were loci encoding members of the ETS family of transcription factors Erg and Ets1, which were the targets for transposons in 87.5% and 65% of the independent JAK2SB^vav leukemias analyzed. ETS proteins are a family of over 20 helix–loop–helix domain transcription factors with diverse biological roles, including important functions in hematopoiesis and leukemia. In normal hematopoiesis, Erg is required for hematopoietic stem cell self-renewal during times of high hematopoietic stem cell cycling (11 ⇓–13). Whereas the ERG locus has been studied extensively in prostate cancer, in which translocation of ERG to the TMPRSS2 locus and expression of a TMPRSS2-ERG fusion oncoprotein occurs in the majority of cases (8), ERG is also known to be translocated to the fused in sarcoma/translocated in liposarcoma (FUS/TLS) locus in rare cases of acute myeloid leukemia (AML). Moreover, increased expression of ERG is associated with poor prognosis in cytogenetically normal AML and T cell acute lymphoblastic leukemia (T-ALL) (7), and the ERG gene resides in the critical region of chromosome 21 that is associated with myeloproliferation and leukemia in Down syndrome (14, 15). Ets-1 is also important for normal hematopoiesis, being involved in the development of both lymphoid and myeloid cell lineages (16). Aberrant expression of ETS1 is implicated in solid tumor development and tumor angiogenesis (17). In hematological malignancy, rare cases of translocation involving ETS1 have been reported (18, 19), and ETS1 may mediate differentiation arrest in T-ALL (20). In addition, the Ets-1 homolog, v-ets, contributes to E26 avian acute leukemia virus-induced myelo-erythroid leukemia in chickens (21).

Analysis of transposon insertion sites within the Erg locus in JAK2SB^vav leukemias revealed, in all cases, intragenic, sense-oriented transposon integration, consistent with activation of Erg via expression of a transposon-Erg fusion transcript, reminiscent of that observed in the ERG intragenic translocations that characterize prostate cancer and AML (7, 8). To validate the role of transposon-mediated activation of Erg in JAK2SB^vav leukemia, we demonstrated that TLS-ERG, a human leukemia-derived fusion gene mimicking intragenic insertion within Erg, induced erythroleukemic pathology in recipients of transduced fetal liver cells. These data complement our previous study in which transduction of fetal liver cells with full-length Erg was also found to induce erythro-megakaryocytic leukemia (22). Of interest, unlike full-length Erg, TLS-ERG did not induce development of lymphoid leukemia, and erythro-megakaryocytic leukemia cells from JAK2SB^vav mice were found to grow more aggressively in culture than their full-length Erg counterparts. These observations suggest subtle differences in the activity of full-length versus truncated Erg alleles.

The TLS-ERG–induced leukemias, like those in JAK2SB^vav mice with Erg and/or Ets1 insertions, were characterized by accumulation of immature erythroid cells, a phenotype also observed in the leukemias driven by full-length Erg (22). Expression of TLS-ERG in human cord blood progenitor cells has been shown to result in a block in erythroid differentiation (23) whereas expression of ERG in K562 cells induces down-regulation of erythroid markers (15), and overexpression of Ets-1 blocks erythroid differentiation in human and mouse hematopoietic progenitor cells (24, 25). Together, these observations suggest that transposon insertion activates the Erg and Ets1 loci and contributes to erythroleukemia by interfering with erythroid differentiation. In this regard, it is noteworthy that the ETS family member Fli-1, although not identified as a CIS in JAK2SB^vav leukemias, is activated via retroviral insertional mutagenesis in friend murine leukemia virus (F-MuLV)–induced erythroleukemias, also blocking erythroid differentiation (26). In over half the JAK2SB^vav leukemias, transposon insertion into both Erg and Ets1 was evident, suggesting that co-occurrence of activation of these ETS family proteins may serve to reinforce the maturation block that contributes to erythroid transformation. In addition to Erg and Ets1, CIS in JAK2SB^vav leukemias were identified at several additional loci with known links to hematological malignancies, including Zmiz1, a gene fusion partner of Abl oncogene 1 (ABL1) (27), Bcor, a transcriptional corepressor mutated in AML (28), and Vav1, commonly deregulated in lymphoid malignancy (29), suggesting that these genes might also contribute to erythroleukemia development.

To identify other loci contributing to erythroid transformation and to specifically explore pathways that cooperate with activation of Erg in disease, the CIS identified in JAK2SB^vav mice were supplemented with CIS in transposon-accelerated TLS-ERG-induced leukemia. Whereas loci encoding sequence-specific DNA binding transcription factors were not identified as CIS in TLS-ERG leukemias, several epigenetic regulators of gene expression emerged, including CREB binding protein (Crebbp), E1A binding protein p300 (Ep300), and enhancer of zeste homolog 2 (Ezh2), which have previously been linked to hematological malignancies (30, 31). Significantly, Jak2 was identified as a CIS in TLS-ERG–driven disease, providing strong validation for cooperation between deregulation of Erg and Jak2 in erythroid transformation. Except for two loci, the CIS identified in JAK2SB^vav and TLS-ERG–induced leukemias were mutually exclusive, presumably reflecting the need for different leukemogenic secondary lesions in erythroid cells already expressing deregulated Jak2 or Erg. Strikingly, classification of CIS in TLS-ERG–driven leukemias into specific pathways or functional classes revealed a particular enrichment for loci expressing regulators of cytokine/growth factor signal transduction pathways, with Jak2 being just one of several such regulators identified, also observed to a lesser extent in JAK2SB^vav leukemias. Thus, accumulation of multiple lesions in signaling pathways may contribute to the evolution of disease. Indeed, analysis of primary JAK2SB^vav leukemias revealed few if any EPO-independent leukemic clones, but these clones began to emerge in the leukemias developing in transplant recipients. Together, these results support a model in which accumulation of lesions inhibiting erythroid maturation, most notably in ETS family members, in combination with mutations that reduce dependence on exogenous cytokine/growth factor signaling, commonly collaborate to drive erythroid transformation in mice. Consistent with this hypothesis, activation of the EPO receptor by spleen focus forming virus (SFFV) glycoprotein gp55 is accompanied by insertional activation of the Ets transcription factor SFFV proviral integration oncogene 1 (Spi1) in SFFV-induced erythroleukemia, and mutations in the gene encoding the SCF receptor, c-Kit, are commonly found in erythroleukemias driven by transgenic expression of Spi1 (26).

The rarity of AEL has contributed to the relative paucity of information on molecular changes associated with human disease. Aneuploidy and complex karyotype, particularly hypodiploidy, are common in AEL and are associated with poor prognosis. Specific genetic changes, however, are poorly defined, with fms-related tyrosine kinase 3 (FLT3) mutations in a small subset of AEL patients, very rare translocations involving JAK2, runt-related transcription factor 1 (RUNX1) mutations in two of a small sample of AELs, and P53 mutations among the few molecular lesions defined (9, 10). Improved diagnosis, prognosis, and treatment of AEL would benefit from an improved understanding of the genetic lesions underlying disease pathogenesis. The suite of genes commonly targeted for transposon-mediated mutagenesis in the murine erythroleukemias described here provide a resource for informing future studies examining genetic changes in human erythroleukemia, as well as a refined list of candidates for systematically dissecting the functional contribution of genetic lesions to erythroleukemia in model systems.

Materials and Methods

Mice.

Mice homozygous for both a T2/Onc transposon array and a cre-inducible (3) or constitutive (32) SB transposase allele and mice hemizygous for transgenes allowing expression of cre (4) and JAK2V617F (5) from the vav1 promoter have been published previously. Animal experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee. For infection of fetal liver cells, virus supernatant was produced in 293T cells as described previously (33) and cells were injected i.v. into mice receiving a single dose of 5.5 Gy γ-irradiation.

Hematological and Histopathological Analysis.

Peripheral blood from the orbital plexus was analyzed by an ADVIA 120 blood analyzer (Bayer). Clonal agar cultures were performed as described (34). Methylcellulose cultures were incubated for up to 14 d at 37 °C in a mixture of 5% (vol/vol) CO₂ in air in Methocult M3234 (Stem Cell Technologies) supplemented with 100 ng/mL SCF, 10 ng/mL IL-3, and 4 IU/mL EPO. For histopathological studies, organs were fixed in 10% (vol/vol) buffered formalin and paraffin embedded. Sections were prepared and stained with hematoxylin and eosin. In transplantation assays, C57BL/6 recipients were injected via the tail vein with 10⁷ cells from spleen or bone marrow of each leukemia-bearing donor, with or without 5.5 Gy γ-irradiation.

DNA Sequencing and Identification of Transposon Integration Sites.

Genomic DNA was extracted from the spleens of leukemic mice using the DNeasy Blood and Tissue Kit (QIAGEN). Enzymatic digestion of DNA and linker-mediated PCR enrichment of transposon junctions were performed following published protocols (35 ⇓–37), with modifications as described in SI Materials and Methods. Identification of common transposon insertion was based on published methods (38) as described in SI Materials and Methods.

Acknowledgments

We thank Janelle Lochland, Jason Corbin, Sheree Brown, Melanie Howell, Lauren Wilkins, and Keti Stoev for skilled assistance and Jelle ten Hoeve (Netherlands Cancer Institute) for making available the unpublished CIMPL software package. This work was supported by Program Grants 1016647 and 490037; fellowships (to W.S.A. and G.K.S.); Independent Research Institutes Infrastructure Support Scheme Grant 361646 from the Australian National Health and Medical Research Council; the Carden Fellowship (to D.M.) of the Cancer Council, Victoria; the Australian Cancer Research Fund; Victorian State Government Operational Infrastructure Support; Cancer Institute New South Wales and Northern Translational Cancer Research Network Fellowships (to V.M.H.); Cure Cancer Australia Foundation and Cancer Council New South Wales project grants; and a Cure Cancer Australia/Leukaemia Foundation Australia Post Doctoral Fellowship and Lions Fellowship, Cancer Council of Victoria (to A.P.N.).

Footnotes

↵¹To whom correspondence may be addressed. E-mail: metcalf{at}wehi.edu.au or alexandw{at}wehi.edu.au.

Author contributions: J.Z.T., C.L.C., W.S., D.M., A.P.N., G.K.S., B.T.K., and W.S.A. designed research; J.Z.T., C.L.C., W.S., D.M., A.P.N., C.D.H., G.K.S., and W.S.A. performed research; N.A.J., N.G.C., V.M.H., and Z.J.Z. contributed new reagents/analytic tools; J.Z.T., C.L.C., W.S., D.M., A.P.N., C.D.H., G.K.S., B.T.K., and W.S.A. analyzed data; and J.Z.T., C.L.C., and W.S.A. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1304234110/-/DCSupplemental.

References

↵
1. Kool J,
2. Berns A
(2009) High-throughput insertional mutagenesis screens in mice to identify oncogenic networks. Nat Rev Cancer 9(6):389–399.
OpenUrl CrossRef PubMed
↵
1. Copeland NG,
2. Jenkins NA
(2010) Harnessing transposons for cancer gene discovery. Nat Rev Cancer 10(10):696–706.
OpenUrl CrossRef PubMed
↵
1. Dupuy AJ,
2. et al.
(2009) A modified sleeping beauty transposon system that can be used to model a wide variety of human cancers in mice. Cancer Res 69(20):8150–8156.
OpenUrl Abstract/FREE Full Text
↵
1. Croker BA,
2. et al.
(2004) SOCS3 is a critical physiological negative regulator of G-CSF signaling and emergency granulopoiesis. Immunity 20(2):153–165.
OpenUrl CrossRef PubMed
↵
1. Xing S,
2. et al.
(2008) Transgenic expression of JAK2V617F causes myeloproliferative disorders in mice. Blood 111(10):5109–5117.
OpenUrl Abstract/FREE Full Text
↵
1. Scott LM
(2011) The JAK2 exon 12 mutations: A comprehensive review. Am J Hematol 86(8):668–676.
OpenUrl CrossRef PubMed
↵
1. Martens JH
(2011) Acute myeloid leukemia: A central role for the ETS factor ERG. Int J Biochem Cell Biol 43(10):1413–1416.
OpenUrl CrossRef PubMed
↵
1. Clark JP,
2. Cooper CS
(2009) ETS gene fusions in prostate cancer. Nat Rev Urol 6(8):429–439.
OpenUrl CrossRef PubMed
↵
1. Santos FP,
2. Bueso-Ramos CE,
3. Ravandi F
(2010) Acute erythroleukemia: Diagnosis and management. Expert Rev Hematol 3(6):705–718.
OpenUrl CrossRef PubMed
↵
1. Zuo Z,
2. Polski JM,
3. Kasyan A,
4. Medeiros LJ
(2010) Acute erythroid leukemia. Arch Pathol Lab Med 134(9):1261–1270.
OpenUrl PubMed
↵
1. Ng AP,
2. et al.
(2011) Erg is required for self-renewal of hematopoietic stem cells during stress hematopoiesis in mice. Blood 118(9):2454–2461.
OpenUrl Abstract/FREE Full Text
↵
1. Loughran SJ,
2. et al.
(2008) The transcription factor Erg is essential for definitive hematopoiesis and the function of adult hematopoietic stem cells. Nat Immunol 9(7):810–819.
OpenUrl CrossRef PubMed
↵
1. Taoudi S,
2. et al.
(2011) ERG dependence distinguishes developmental control of hematopoietic stem cell maintenance from hematopoietic specification. Genes Dev 25(3):251–262.
OpenUrl Abstract/FREE Full Text
↵
1. Ng AP,
2. et al.
(2010) Trisomy of Erg is required for myeloproliferation in a mouse model of Down syndrome. Blood 115(19):3966–3969.
OpenUrl Abstract/FREE Full Text
↵
1. Rainis L,
2. et al.
(2005) The proto-oncogene ERG in megakaryoblastic leukemias. Cancer Res 65(17):7596–7602.
OpenUrl Abstract/FREE Full Text
↵
1. Bories JC,
2. et al.
(1995) Increased T-cell apoptosis and terminal B-cell differentiation induced by inactivation of the Ets-1 proto-oncogene. Nature 377(6550):635–638.
OpenUrl CrossRef PubMed
↵
1. Hahne JC,
2. et al.
(2008) The transcription factor ETS-1: Its role in tumour development and strategies for its inhibition. Mini Rev Med Chem 8(11):1095–1105.
OpenUrl CrossRef PubMed
↵
1. Goyns MH,
2. Hann IM,
3. Stewart J,
4. Gegonne A,
5. Birnie GD
(1987) The c-ets-1 proto-oncogene is rearranged in some cases of acute lymphoblastic leukaemia. Br J Cancer 56(5):611–613.
OpenUrl CrossRef PubMed
↵
1. Rovigatti U,
2. Watson DK,
3. Yunis JJ
(1986) Amplification and rearrangement of Hu-ets-1 in leukemia and lymphoma with involvement of 11q23. Science 232(4748):398–400.
OpenUrl Abstract/FREE Full Text
↵
1. Dadi S,
2. et al.
(2012) TLX homeodomain oncogenes mediate T cell maturation arrest in T-ALL via interaction with ETS1 and suppression of TCRα gene expression. Cancer Cell 21(4):563–576.
OpenUrl CrossRef PubMed
↵
1. Metz T,
2. Graf T
(1991) v-myb and v-ets transform chicken erythroid cells and cooperate both in trans and in cis to induce distinct differentiation phenotypes. Genes Dev 5(3):369–380.
OpenUrl Abstract/FREE Full Text
↵
1. Carmichael CL,
2. et al.
(2012) Hematopoietic overexpression of the transcription factor Erg induces lymphoid and erythro-megakaryocytic leukemia. Proc Natl Acad Sci USA 109(38):15437–15442.
OpenUrl Abstract/FREE Full Text
↵
1. Pereira DS,
2. et al.
(1998) Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells. Proc Natl Acad Sci USA 95(14):8239–8244.
OpenUrl Abstract/FREE Full Text
↵
1. Lulli V,
2. et al.
(2006) Overexpression of Ets-1 in human hematopoietic progenitor cells blocks erythroid and promotes megakaryocytic differentiation. Cell Death Differ 13(7):1064–1074.
OpenUrl CrossRef PubMed
↵
1. Marziali G,
2. et al.
(2002) Role of Ets-1 in erythroid differentiation. Blood Cells Mol Dis 29(3):553–561.
OpenUrl CrossRef PubMed
↵
1. Moreau-Gachelin F
(2008) Multi-stage Friend murine erythroleukemia: molecular insights into oncogenic cooperation. Retrovirology 5:99.
OpenUrl CrossRef PubMed
↵
1. De Braekeleer E,
2. et al.
(2011) ABL1 fusion genes in hematological malignancies: A review. Eur J Haematol 86(5):361–371.
OpenUrl CrossRef PubMed
↵
1. Grossmann V,
2. et al.
(2011) Whole-exome sequencing identifies somatic mutations of BCOR in acute myeloid leukemia with normal karyotype. Blood 118(23):6153–6163.
OpenUrl Abstract/FREE Full Text
↵
1. Oberley MJ,
2. Wang DS,
3. Yang DT
(2012) Vav1 in hematologic neoplasms, a mini review. Am J Blood Res 2(1):1–8.
OpenUrl PubMed
↵
1. Shih AH,
2. Abdel-Wahab O,
3. Patel JP,
4. Levine RL
(2012) The role of mutations in epigenetic regulators in myeloid malignancies. Nat Rev Cancer 12(9):599–612.
OpenUrl CrossRef PubMed
↵
1. Shima Y,
2. Kitabayashi I
(2011) Deregulated transcription factors in leukemia. Int J Hematol 94(2):134–141.
OpenUrl CrossRef PubMed
↵
1. Dupuy AJ,
2. Akagi K,
3. Largaespada DA,
4. Copeland NG,
5. Jenkins NA
(2005) Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436(7048):221–226.
OpenUrl CrossRef PubMed
↵
1. Glaser SP,
2. et al.
(2012) Anti-apoptotic Mcl-1 is essential for the development and sustained growth of acute myeloid leukemia. Genes Dev 26(2):120–125.
OpenUrl Abstract/FREE Full Text
↵
1. Alexander WS,
2. Roberts AW,
3. Nicola NA,
4. Li R,
5. Metcalf D
(1996) Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietic receptor c-Mpl. Blood 87(6):2162–2170.
OpenUrl Abstract/FREE Full Text
↵
1. Keng VW,
2. et al.
(2009) A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma. Nat Biotechnol 27(3):264–274.
OpenUrl CrossRef PubMed
↵
1. Largaespada DA,
2. Collier LS
(2008) Transposon-mediated mutagenesis in somatic cells: Identification of transposon-genomic DNA junctions. Methods Mol Biol 435:95–108.
OpenUrl CrossRef PubMed
↵
1. Starr TK,
2. et al.
(2009) A transposon-based genetic screen in mice identifies genes altered in colorectal cancer. Science 323(5922):1747–1750.
OpenUrl Abstract/FREE Full Text
↵
1. March HN,
2. et al.
(2011) Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis. Nat Genet 43(12):1202–1209.
OpenUrl CrossRef PubMed

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Proceedings of the National Academy of Sciences: 110 (15)

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[1] ↵
Kool J,
Berns A
(2009) High-throughput insertional mutagenesis screens in mice to identify oncogenic networks. Nat Rev Cancer 9(6):389–399.
OpenUrl CrossRef PubMed

[2] Kool J,

[3] Berns A

[4] ↵
Copeland NG,
Jenkins NA
(2010) Harnessing transposons for cancer gene discovery. Nat Rev Cancer 10(10):696–706.
OpenUrl CrossRef PubMed

[5] Copeland NG,

[6] Jenkins NA

[7] ↵
Dupuy AJ,
et al.
(2009) A modified sleeping beauty transposon system that can be used to model a wide variety of human cancers in mice. Cancer Res 69(20):8150–8156.
OpenUrl Abstract/FREE Full Text

[8] Dupuy AJ,

[9] et al.

[10] ↵
Croker BA,
et al.
(2004) SOCS3 is a critical physiological negative regulator of G-CSF signaling and emergency granulopoiesis. Immunity 20(2):153–165.
OpenUrl CrossRef PubMed

[11] Croker BA,

[12] et al.

[13] ↵
Xing S,
et al.
(2008) Transgenic expression of JAK2V617F causes myeloproliferative disorders in mice. Blood 111(10):5109–5117.
OpenUrl Abstract/FREE Full Text

[14] Xing S,

[15] et al.

[16] ↵
Scott LM
(2011) The JAK2 exon 12 mutations: A comprehensive review. Am J Hematol 86(8):668–676.
OpenUrl CrossRef PubMed

[17] Scott LM

[18] ↵
Martens JH
(2011) Acute myeloid leukemia: A central role for the ETS factor ERG. Int J Biochem Cell Biol 43(10):1413–1416.
OpenUrl CrossRef PubMed

[19] Martens JH

[20] ↵
Clark JP,
Cooper CS
(2009) ETS gene fusions in prostate cancer. Nat Rev Urol 6(8):429–439.
OpenUrl CrossRef PubMed

[21] Clark JP,

[22] Cooper CS

[23] ↵
Santos FP,
Bueso-Ramos CE,
Ravandi F
(2010) Acute erythroleukemia: Diagnosis and management. Expert Rev Hematol 3(6):705–718.
OpenUrl CrossRef PubMed

[24] Santos FP,

[25] Bueso-Ramos CE,

[26] Ravandi F

[27] ↵
Zuo Z,
Polski JM,
Kasyan A,
Medeiros LJ
(2010) Acute erythroid leukemia. Arch Pathol Lab Med 134(9):1261–1270.
OpenUrl PubMed

[28] Zuo Z,

[29] Polski JM,

[30] Kasyan A,

[31] Medeiros LJ

[32] ↵
Ng AP,
et al.
(2011) Erg is required for self-renewal of hematopoietic stem cells during stress hematopoiesis in mice. Blood 118(9):2454–2461.
OpenUrl Abstract/FREE Full Text

[33] Ng AP,

[34] et al.

[35] ↵
Loughran SJ,
et al.
(2008) The transcription factor Erg is essential for definitive hematopoiesis and the function of adult hematopoietic stem cells. Nat Immunol 9(7):810–819.
OpenUrl CrossRef PubMed

[36] Loughran SJ,

[37] et al.

[38] ↵
Taoudi S,
et al.
(2011) ERG dependence distinguishes developmental control of hematopoietic stem cell maintenance from hematopoietic specification. Genes Dev 25(3):251–262.
OpenUrl Abstract/FREE Full Text

[39] Taoudi S,

[40] et al.

[41] ↵
Ng AP,
et al.
(2010) Trisomy of Erg is required for myeloproliferation in a mouse model of Down syndrome. Blood 115(19):3966–3969.
OpenUrl Abstract/FREE Full Text

[42] Ng AP,

[43] et al.

[44] ↵
Rainis L,
et al.
(2005) The proto-oncogene ERG in megakaryoblastic leukemias. Cancer Res 65(17):7596–7602.
OpenUrl Abstract/FREE Full Text

[45] Rainis L,

[46] et al.

[47] ↵
Bories JC,
et al.
(1995) Increased T-cell apoptosis and terminal B-cell differentiation induced by inactivation of the Ets-1 proto-oncogene. Nature 377(6550):635–638.
OpenUrl CrossRef PubMed

[48] Bories JC,

[49] et al.

[50] ↵
Hahne JC,
et al.
(2008) The transcription factor ETS-1: Its role in tumour development and strategies for its inhibition. Mini Rev Med Chem 8(11):1095–1105.
OpenUrl CrossRef PubMed

[51] Hahne JC,

[52] et al.

[53] ↵
Goyns MH,
Hann IM,
Stewart J,
Gegonne A,
Birnie GD
(1987) The c-ets-1 proto-oncogene is rearranged in some cases of acute lymphoblastic leukaemia. Br J Cancer 56(5):611–613.
OpenUrl CrossRef PubMed

[54] Goyns MH,

[55] Hann IM,

[56] Stewart J,

[57] Gegonne A,

[58] Birnie GD

[59] ↵
Rovigatti U,
Watson DK,
Yunis JJ
(1986) Amplification and rearrangement of Hu-ets-1 in leukemia and lymphoma with involvement of 11q23. Science 232(4748):398–400.
OpenUrl Abstract/FREE Full Text

[60] Rovigatti U,

[61] Watson DK,

[62] Yunis JJ

[63] ↵
Dadi S,
et al.
(2012) TLX homeodomain oncogenes mediate T cell maturation arrest in T-ALL via interaction with ETS1 and suppression of TCRα gene expression. Cancer Cell 21(4):563–576.
OpenUrl CrossRef PubMed

[64] Dadi S,

[65] et al.

[66] ↵
Metz T,
Graf T
(1991) v-myb and v-ets transform chicken erythroid cells and cooperate both in trans and in cis to induce distinct differentiation phenotypes. Genes Dev 5(3):369–380.
OpenUrl Abstract/FREE Full Text

[67] Metz T,

[68] Graf T

[69] ↵
Carmichael CL,
et al.
(2012) Hematopoietic overexpression of the transcription factor Erg induces lymphoid and erythro-megakaryocytic leukemia. Proc Natl Acad Sci USA 109(38):15437–15442.
OpenUrl Abstract/FREE Full Text

[70] Carmichael CL,

[71] et al.

[72] ↵
Pereira DS,
et al.
(1998) Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells. Proc Natl Acad Sci USA 95(14):8239–8244.
OpenUrl Abstract/FREE Full Text

[73] Pereira DS,

[74] et al.

[75] ↵
Lulli V,
et al.
(2006) Overexpression of Ets-1 in human hematopoietic progenitor cells blocks erythroid and promotes megakaryocytic differentiation. Cell Death Differ 13(7):1064–1074.
OpenUrl CrossRef PubMed

[76] Lulli V,

[77] et al.

[78] ↵
Marziali G,
et al.
(2002) Role of Ets-1 in erythroid differentiation. Blood Cells Mol Dis 29(3):553–561.
OpenUrl CrossRef PubMed

[79] Marziali G,

[80] et al.

[81] ↵
Moreau-Gachelin F
(2008) Multi-stage Friend murine erythroleukemia: molecular insights into oncogenic cooperation. Retrovirology 5:99.
OpenUrl CrossRef PubMed

[82] Moreau-Gachelin F

[83] ↵
De Braekeleer E,
et al.
(2011) ABL1 fusion genes in hematological malignancies: A review. Eur J Haematol 86(5):361–371.
OpenUrl CrossRef PubMed

[84] De Braekeleer E,

[85] et al.

[86] ↵
Grossmann V,
et al.
(2011) Whole-exome sequencing identifies somatic mutations of BCOR in acute myeloid leukemia with normal karyotype. Blood 118(23):6153–6163.
OpenUrl Abstract/FREE Full Text

[87] Grossmann V,

[88] et al.

[89] ↵
Oberley MJ,
Wang DS,
Yang DT
(2012) Vav1 in hematologic neoplasms, a mini review. Am J Blood Res 2(1):1–8.
OpenUrl PubMed

[90] Oberley MJ,

[91] Wang DS,

[92] Yang DT

[93] ↵
Shih AH,
Abdel-Wahab O,
Patel JP,
Levine RL
(2012) The role of mutations in epigenetic regulators in myeloid malignancies. Nat Rev Cancer 12(9):599–612.
OpenUrl CrossRef PubMed

[94] Shih AH,

[95] Abdel-Wahab O,

[96] Patel JP,

[97] Levine RL

[98] ↵
Shima Y,
Kitabayashi I
(2011) Deregulated transcription factors in leukemia. Int J Hematol 94(2):134–141.
OpenUrl CrossRef PubMed

[99] Shima Y,

[100] Kitabayashi I

[101] ↵
Dupuy AJ,
Akagi K,
Largaespada DA,
Copeland NG,
Jenkins NA
(2005) Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436(7048):221–226.
OpenUrl CrossRef PubMed

[102] Dupuy AJ,

[103] Akagi K,

[104] Largaespada DA,

[105] Copeland NG,

[106] Jenkins NA

[107] ↵
Glaser SP,
et al.
(2012) Anti-apoptotic Mcl-1 is essential for the development and sustained growth of acute myeloid leukemia. Genes Dev 26(2):120–125.
OpenUrl Abstract/FREE Full Text

[108] Glaser SP,

[109] et al.

[110] ↵
Alexander WS,
Roberts AW,
Nicola NA,
Li R,
Metcalf D
(1996) Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietic receptor c-Mpl. Blood 87(6):2162–2170.
OpenUrl Abstract/FREE Full Text

[111] Alexander WS,

[112] Roberts AW,

[113] Nicola NA,

[114] Li R,

[115] Metcalf D

[116] ↵
Keng VW,
et al.
(2009) A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma. Nat Biotechnol 27(3):264–274.
OpenUrl CrossRef PubMed

[117] Keng VW,

[118] et al.

[119] ↵
Largaespada DA,
Collier LS
(2008) Transposon-mediated mutagenesis in somatic cells: Identification of transposon-genomic DNA junctions. Methods Mol Biol 435:95–108.
OpenUrl CrossRef PubMed

[120] Largaespada DA,

[121] Collier LS

[122] ↵
Starr TK,
et al.
(2009) A transposon-based genetic screen in mice identifies genes altered in colorectal cancer. Science 323(5922):1747–1750.
OpenUrl Abstract/FREE Full Text

[123] Starr TK,

[124] et al.

[125] ↵
March HN,
et al.
(2011) Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis. Nat Genet 43(12):1202–1209.
OpenUrl CrossRef PubMed

[126] March HN,

[127] et al.

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Transposon mutagenesis reveals cooperation of ETS family transcription factors with signaling pathways in erythro-megakaryocytic leukemia

Abstract

Results

JAK2V617F Accelerates SB-Induced Erythroleukemia.

JAK2SB^vav Leukemia Is Transplantable.

Common Transposon Insertion Sites in JAK2SB^vav Leukemia.

Identification of Cooperating Lesions in ERG-Driven Leukemia.

Discussion

Materials and Methods

Mice.

Hematological and Histopathological Analysis.

DNA Sequencing and Identification of Transposon Integration Sites.

Acknowledgments

Footnotes

References

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Abstract

Results

JAK2V617F Accelerates SB-Induced Erythroleukemia.

JAK2SBvav Leukemia Is Transplantable.

Common Transposon Insertion Sites in JAK2SBvav Leukemia.

Identification of Cooperating Lesions in ERG-Driven Leukemia.

Discussion

Materials and Methods

Mice.

Hematological and Histopathological Analysis.

DNA Sequencing and Identification of Transposon Integration Sites.

Acknowledgments

Footnotes

References

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JAK2SB^vav Leukemia Is Transplantable.

Common Transposon Insertion Sites in JAK2SB^vav Leukemia.